Phosphorus

Toxicity to soil macroorganisms except arthropods

Administrative data

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-02-26 to 2010-04-26

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Version / remarks:

DIN ISO 11268-2: 1998 (E), BBA Guideline (1994)

Deviations:

yes

Remarks:

The soil moisture of all test vessels deviated by more than 10 % of the individual at the end of the test since it was decided to add additional water during the study due to a dry soil surface.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Phosphorus

EC Number:

231-768-7

EC Name:

Phosphorus

Cas Number:

7723-14-0

IUPAC Name:

phosphine

Details on test material:

- Name of test material (as cited in study report): Red Phosphorus, untreated
- Molecular weight: Polymeric
- Physical state: Solid, dark reddish brown solid powder
- Analytical purity: 99.2 % (w/w)
- Purity test date: Not specified
- Lot/batch No.: Fra 07-23
- Expiration date of the lot/batch: 2011-04-24
- Stability under test conditions: Not specified
- Storage condition of test material: Room temperature, protected from moisture and light, under nitrogen gas to prevent oxidising.

Sampling and analysis

Analytical monitoring:

no

Test substrate

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): The test item amount for each test item concentration was weighed out and mixed thoroughly with a subsample of moistened artificial soil (10 g dry weight per replicate). The treated soil subsample was given to the artificial soil, which was moistened with 432 g demineralised water to a moisture of 56 % of the maximum water holding capacity.
Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 600 g soil dry weight was filled into each test vessel.
- Controls: Artificial soil moistened with demineralised water without test item was used as control medium.

Test organisms

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Eisenia fetida SAVIGNY 1826, subspecies Eisenia fetida fetida
- Source: Own breeding
- Age at test initiation (mean and range, SD): synchronized earthworm culture, not deviating more than 1 month
- Weight at test initiation (mean and range, SD): 0.50


ACCLIMATION
- Acclimation period: 2 days prior to test start
- Acclimation conditions (same as test or not): same as test
- Health during acclimation (any mortality observed): healthy

Study design

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Remarks:

Removement of adults after 28 days

Test conditions

Test temperature:

18 - 22 °C

pH:

Test item concentration [mg test item/kg soil dry weight] pH-value
day 0 day 56
Control 6.77 6.82
62.5 6.76 6.73
125 6.77 6.78
250 6.76 6.63
500 6.60 6.54
1000 6.57 6.38

Moisture:

Test item concentration [mg test item/kg soil dry weight] Moisture [%] of dry weight

day 0 day 56
Control 33.5 36.4
62.5 32.1 38.9*
125 33.2 39.2*
250 31.9 38.0*
500 30.7 38.9*
1000 31.0 37.6*

Details on test conditions:

TEST SYSTEM
- Test container (material, size): round plastic boxes, ID: 15 cm, bottom surface area: 177 cm3, height: 14 cm, transparent and perforated lids enabled sufficient gas exchange and light penetration and prevented the test substrate from drying
- Amount of soil or substrate: 600 g DW
- No. of organisms per container (treatment): 10 (40)
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control:


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 10 % peat, 20 % kaolin, 69 % air dried quartz sand, 0.35 % calcium carbonate
- Organic carbon (%): not determined
- Maximum water holding capacity (in % dry weigth): 61
- CEC: not determined
- Pretreatment of soil: none
- Storage (condition, duration): 8 days after preparation at test conditions in the dark



OTHER TEST CONDITIONS
- Photoperiod: 16 h light - 8 h dark
- Light intensity: 504 +/- 56 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Adult mortality, biomass, behaviour and pathological symptoms after 28 days
Reproduction (Number of juveniles) after 56 days

VEHICLE CONTROL PERFORMED: not necessary


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

Nominal and measured concentrations:

Nominal application rates: 62.5 - 125 - 250 - 500 - 1000 mg test substance/kg soil dry weight (DW)

Reference substance (positive control):

yes

Remarks:

1.0 - 2.5 - 5.0 mg Carbendazim/ kg soil DW

Results and discussion

Effect concentrations

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

428 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: (342-551)

Details on results:

- Mortality at end of exposure period: One death adult earthworm was found at the test item concentration of 125 mg/kg soil dry weight after 28 days of exposure to the test item. This level of earthworm mortality (2.5 % at 125 mg test item/kg soil dry weight) is not considered to be of biological significance.
- Changes in body weigth of live adults (% of initial weight) at end of exposure period: At test start the earthworms had an individual weight of 0.30 - 0.60 g. The body weights of the adult earthworms increased throughout the first 28 days at the test item concentrations of 62.5 and 125 mg test item/kg soil dry weight without any statistically significant differences compared to the control. However, at 250, 500 and 1000 mg/kg soil dry weight the biomass increase of adult earthworm was statistically significantly reduced compared to the control
- No. of offspring produced: The mean number of juveniles in the control was 280. The average number of juveniles in the treated groups ranged between 31 and 255. Compared to the control the reproduction of the earthworms was not statistically significantly reduced at 62.5 and 125 mg test item/kg soil dry weight. However, at 250, 500 and 1000 mg test item/kg soil dry weight the earthworm reproduction rate was statistically significantly reduced compared to the control.
- Behavioural abnormalities: No noteworthy symptoms or changes in the behaviour of the earthworms were observed in the control and at the test item concentrations of 62.5, 125 and 250 mg test item/kg soil dry weight after 28 days of exposure to Red Phosphorus, untreated. At the test item concentrations of 500 and 1000 mg test item/kg soil dry weight biologically significant symptoms of 17.5 % at 500 mg/kg soil dry weight and of 22.5 % at 1000 mg/kg soil dry weight occurred.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: 2.5 mg Carbendazim/kg soil DW

Reported statistics and error estimates:

One Way Analysis of Variance (ANOVA) was carried out for the determination of statistically significant differences compared to the control. A Normality Test and an Equal Variance Test were conducted prior to running the ANOVA. P-values for both Normality and Equal Variance Test are 0.05. The a-value for ANOVA test (acceptable probability of incorrectly concluding that there is a difference) is a = 0.05. Significant differences (p ≤ 0.05) were determined by using a Dunnett’s test. Since the Equal Variance Test for reproduction failed the data were log10- transformed prior to ANOVA.
The EC50-value for reproduction was calculated by a sigmoidal dose-response regression. Calculation of the confidence interval for EC50-value was carried out.

Any other information on results incl. tables

Mortality of Adult Earthworms in [%] after 28 Days of Exposure

Test item concentration [mg test item/kg soil dry weight]	Mortality [%]
	Replicate				MV
	1	2	3	4
Control	0	0	0	0	0
62.5	0	0	0	0	0
125	0	0	10	0	2.5
250	0	0	0	0	0
500	0	0	0	0	0
1000	0	0	0	0	0

Earthworm Behaviour and Pathological Symptoms after 28 Days of Exposure

Test item concentration    [mg/kg soil dry weight]	Effect	Replicate
		1	2	3	4
Control	A	10/10	10/10	10/10	9/10
	F	–	–	–	1/10
62.5	A	10/10	10/10	10/10	10/10
125	A	10/10	10/10	9/10	10/10
	H	–	–	1/10	–
250	A	9/10	10/10	10/10	9/10
	F	1/10	–	–	–
	G	–	–	–	1/10
500	A	10/10	7/10	10/10	6/10
	D	–	2/10	–	–
	G	–	1/10	–	4/10
1000	A	8/10	8/10	8/10	7/10
	G	2/10	2/10	2/10	3/10

Body Weight Changes of the Adult Earthworms      

Test item concentration [mg test item/kg soil dry weight]	Rep.	Mean body weights per replicate		Mean body weight change of earthworms
		Test start	28 days	Per replicate		Mean ± SD		Sign.*
		[g]	[g]	[g]	[%]	[g]	[%]
Control	1	0.457	0.640	0.18	40.0	0.18 ± 0.02	36.2 ± 3.79	–
	2	0.522	0.725	0.20	38.9
	3	0.534	0.710	0.18	33.0
	4	0.504	0.670	0.17	32.9
62.5	1	0.515	0.700	0.19	35.9	0.17 ± 0.04	32.7 ± 6.06	No
	2	0.464	0.584	0.12	25.9
	3	0.504	0.653	0.15	29.6
	4	0.532	0.741	0.21	39.3
125	1	0.529	0.637	0.11	20.4	0.12 ± 0.08	24.8 ± 16.3	No
	2	0.500	0.670	0.17	34.0
	3	0.459	0.647	0.19	40.9
	4	0.516	0.536	0.02	3.88
250	1	0.519	0.592	0.07	14.1	0.09 ± 0.03	17.6 + 5.90	Yes
	2	0.529	0.632	0.10	19.5
	3	0.503	0.562	0.06	11.7
	4	0.461	0.576	0.12	24.9
500	1	0.461	0.472	0.01	2.39	0.00 ± 0.04	-0.12 ± 6.95	Yes
	2	0.521	0.467	-0.05	-10.4
	3	0.530	0.557	0.03	5.09
	4	0.499	0.511	0.01	2.40
1000	1	0.526	0.502	-0.02	-4.56	-0.01 ± 0.02	-2.06 ± 4.71	Yes
	2	0.464	0.480	0.02	3.45
	3	0.504	0.468	-0.04	-7.14
	4	0.515	0.515	0.00	0.00

Reproduction Rate (Number of Juveniles after 8 Weeks)

Test item concentration [mg test item/kg soil dry weight]	Rep.	Number of juveniles	Mean ± SD	CV               [%]	[%] of control	Significance compared to control
Control	1	289	280 ± 26.0	9.29	–	–
	2	245
	3	280
	4	307
62.5	1	212	255 ± 41.4	16.3	90.8	No
	2	289
	3	291
	4	226
125	1	286	240 ± 46.8	19.5	85.5	No
	2	265
	3	229
	4	179
250	1	220	203 ± 32.6	16.1	72.4	Yes
	2	179
	3	172
	4	240
500	1	109	118 ± 12.2	10.3	42.1	Yes
	2	108
	3	134
	4	121
1000	1	40	30.5 ± 7.33	24.0	10.9	Yes
	2	32
	3	23
	4	27

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, Red Phosphorus, untreated did not induce evident mortality of adult earthworms after 28 days of exposure. No pathological symptoms or changes of the behaviour of adult earthworms were observed at 62.5, 125 and 250 mg test item/kg soil dry weight. However, at 500 and 1000 mg test item/kg soil dry weight biologically significant symptoms occurred. The earthworm biomass increased in the control and at 62.5 and 125 mg test item/kg soil dry weight over 28 days without any statistically significant differences. At 250, 500 and 1000 mg test item/kg soil dry weight the biomass increase was statistically significantly reduced. After eight weeks of exposure the earthworm reproduction did not statistically significantly differ to the control at 62.5 and 125 mg test item/kg soil dry weight. However, at 250, 500 and 1000 mg test item/kg soil dry the reproduction rate of adult earthworms was statistically significantly reduced compared to the control.

Thus the No Observed Effect Concentration (NOEC) for Red Phosphorus, untreated with respect to mortality, body weight and reproduction of Eisenia fetida fetida after 8 weeks of exposure in artificial soil was determined to be at 125 mg test item/kg soil dry weight.

Executive summary:

Effects of Red Phosphorus, untreated (Batch number: Fra 07-23) on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Savigny) were determined according to the guidelines DIN ISO 11268-2 (1998), OECD 222 (2004) and BBA (1994) from February 26^thto April 26^th, 2010 with the definitive exposure phase from February 26^th to April 23^rd, 2010 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany. The study was conducted under static conditions over 8 weeks with the test item concentrations of 62.5, 125, 250, 500 and 1000 mg test item/kg soil dry weight, which were mixed into artificial soil containing 10 % peat and a control using untreated artificial soil. 40 test organisms, with an individual weight of 0.30 to 0.60 g, were divided into 4 replicate groups with 10 test organisms per replicate.

After 28 days no evident mortality of adult earthworms was observed in the control and at all test item concentrations. No pathological symptoms or changes in the behaviour of adult earthworms were seen in the control as well as at 62.5, 125 and 250 mg test item/kg soil dry weight. However, at 500 and 1000 mg test item/kg soil dry weight biologically significant pathological symptoms of the adult earthworms occurred. The earthworm biomass increased in the control and at 62.5 and 125 mg test item/kg soil dry weight over 28 days without any statistically significant differences. At 250, 500 and 1000 mg test item/kg soil dry weight the biomass increase was statistically significantly reduced compared to the control.

After further four weeks the reproduction rate (average number of juveniles) was 280 in the control and ranged between 31 and 255 at the test item concentrations. Compared to the control, the reproduction rate was statistically significantly reduced at 250, 500 and 1000 mg test item/kg soil dry weight .The EC₅₀ for reproduction was determined to be at 428 mg/kg soil dry weight. 

Overall, the NOEC of the test item concerning mortality, biomass and reproduction of earthworms was determined to be at 125 mg test item/kg soil dry weight.

All validity criteria recommended by the test guidelines were fulfilled.

Summary of All Observed Effects in the Tested Doses

Effects	Red Phosphorus, untreated
[mg test item/kg soil dry weight]	62.5	125	250	500	1000
Mortality of adult earthworms	No	No	No	No	No
Reduction of body weight	No	No	Yes	Yes	Yes
Reduced reproduction rate   (Number of juveniles)	No	No	Yes	Yes	Yes
Summary of all effects	No	No	Yes	Yes	Yes

NOEC and LOEC for Reproduction, Mortality and Body Weight

Red Phosphorus, untreated
	Test item concentration
	[mg test item/kg soil dry weight]
NOECreproduction, body weight	125
LOECreproduction, body weight	250
NOEC mortality	1000
LOEC mortality	> 1000
EC50  of reproduction (95 % Confidence Interval)	428 (342 - 551)